Molecular basis of FAAH-OUT-associated human pain insensitivity.

Chronic pain affects millions of people worldwide and new treatments are needed urgently. One way to identify novel analgesic strategies is to understand the biological dysfunctions that lead to human inherited pain insensitivity disorders. Here we report how the recently discovered brain and dorsal root ganglia-expressed FAAH-OUT long non-coding RNA (lncRNA) gene, which was found from studying a pain-insensitive patient with reduced anxiety and fast wound healing, regulates the adjacent key endocannabinoid system gene FAAH, which encodes the anandamide-degrading fatty acid amide hydrolase enzyme. We demonstrate that the disruption in FAAH-OUT lncRNA transcription leads to DNMT1-dependent DNA methylation within the FAAH promoter. In addition, FAAH-OUT contains a conserved regulatory element, FAAH-AMP, that acts as an enhancer for FAAH expression. Furthermore, using transcriptomic analyses in patient-derived cells we have uncovered a network of genes that are dysregulated from disruption of the FAAH-FAAH-OUT axis, thus providing a coherent mechanistic basis to understand the human phenotype observed. Given that FAAH is a potential target for the treatment of pain, anxiety, depression and other neurological disorders, this new understanding of the regulatory role of the FAAH-OUT gene provides a platform for the development of future gene and small molecule therapies.

Pregabalin Silences Oxaliplatin-Activated Sensory Neurons to Relieve Cold Allodynia.

Oxaliplatin is a platinum-based chemotherapeutic agent that causes cold and mechanical allodynia in up to 90% of patients. Silent Nav1.8-positive nociceptive cold sensors have been shown to be unmasked by oxaliplatin, and this event has been causally linked to the development of cold allodynia. We examined the effects of pregabalin on oxaliplatin-evoked unmasking of cold sensitive neurons using mice expressing GCaMP-3 in all sensory neurons. Intravenous injection of pregabalin significantly ameliorates cold allodynia, while decreasing the number of cold sensitive neurons by altering their excitability and temperature thresholds. The silenced neurons are predominantly medium/large mechano-cold sensitive neurons, corresponding to the "silent" cold sensors activated during neuropathy. Deletion of α2δ1 subunits abolished the effects of pregabalin on both cold allodynia and the silencing of sensory neurons. Thus, these results define a novel, peripheral inhibitory effect of pregabalin on the excitability of "silent" cold-sensing neurons in a model of oxaliplatin-dependent cold allodynia.

A central mechanism of analgesia in mice and humans lacking the sodium channel NaV1.7.

Deletion of SCN9A encoding the voltage-gated sodium channel NaV1.7 in humans leads to profound pain insensitivity and anosmia. Conditional deletion of NaV1.7 in sensory neurons of mice also abolishes pain, suggesting that the locus of analgesia is the nociceptor. Here we demonstrate, using in vivo calcium imaging and extracellular recording, that NaV1.7 knockout mice have essentially normal nociceptor activity. However, synaptic transmission from nociceptor central terminals in the spinal cord is greatly reduced by an opioid-dependent mechanism. Analgesia is also reversed substantially by central but not peripheral application of opioid antagonists. In contrast, the lack of neurotransmitter release from olfactory sensory neurons is opioid independent. Male and female humans with NaV1.7-null mutations show naloxone-reversible analgesia. Thus, inhibition of neurotransmitter release is the principal mechanism of anosmia and analgesia in mouse and human Nav1.7-null mutants.

Silent cold-sensing neurons contribute to cold allodynia in neuropathic pain.

Patients with neuropathic pain often experience innocuous cooling as excruciating pain. The cell and molecular basis of this cold allodynia is little understood. We used in vivo calcium imaging of sensory ganglia to investigate how the activity of peripheral cold-sensing neurons was altered in three mouse models of neuropathic pain: oxaliplatin-induced neuropathy, partial sciatic nerve ligation, and ciguatera poisoning. In control mice, cold-sensing neurons were few in number and small in size. In neuropathic animals with cold allodynia, a set of normally silent large diameter neurons became sensitive to cooling. Many of these silent cold-sensing neurons responded to noxious mechanical stimuli and expressed the nociceptor markers Nav1.8 and CGRPα. Ablating neurons expressing Nav1.8 resulted in diminished cold allodynia. The silent cold-sensing neurons could also be activated by cooling in control mice through blockade of Kv1 voltage-gated potassium channels. Thus, silent cold-sensing neurons are unmasked in diverse neuropathic pain states and cold allodynia results from peripheral sensitization caused by altered nociceptor excitability.

Tools for analysis and conditional deletion of subsets of sensory neurons.

Background: Somatosensation depends on primary sensory neurons of the trigeminal and dorsal root ganglia (DRG). Transcriptional profiling of mouse DRG sensory neurons has defined at least 18 distinct neuronal cell types. Using an advillin promoter, we have generated a transgenic mouse line that only expresses diphtheria toxin A (DTA) in sensory neurons in the presence of Cre recombinase. This has allowed us to ablate specific neuronal subsets within the DRG using a range of established and novel Cre lines that encompass all sets of sensory neurons.    Methods: A floxed-tdTomato-stop-DTA bacterial artificial chromosome (BAC) transgenic reporter line (AdvDTA) under the control of the mouse advillin DRG promoter was generated. The line was first validated using a Na v1.8 Cre and then crossed to CGRP CreER (Calca), Th CreERT2, Tmem45b Cre, Tmem233 Cre, Ntng1 Cre and TrkB CreER (Ntrk2) lines. Pain behavioural assays included Hargreaves', hot plate, Randall-Selitto, cold plantar, partial sciatic nerve ligation and formalin tests. Results: Motor activity, as assessed by the rotarod test, was normal for all lines tested. Noxious mechanosensation was significantly reduced when either Na v1.8 positive neurons or Tmem45b positive neurons were ablated whilst acute heat pain was unaffected. In contrast, noxious mechanosensation was normal following ablation of CGRP-positive neurons but acute heat pain thresholds were significantly elevated and a reduction in nocifensive responses was observed in the second phase of the formalin test. Ablation of TrkB-positive neurons led to significant deficits in mechanical hypersensitivity in the partial sciatic nerve ligation neuropathic pain model. Conclusions: Ablation of specific DRG neuronal subsets using the AdvDTA line will be a useful resource for further functional characterization of somatosensory processing, neuro-immune interactions and chronic pain disorders.

Sensitization of Cutaneous Primary Afferents in Bone Cancer Revealed by In Vivo Calcium Imaging.

Cancer-induced bone pain (CIBP) is a complex condition, comprising components of inflammatory and neuropathic processes, but changes in the physiological response profiles of bone-innervating and cutaneous afferents remain poorly understood. We used a combination of retrograde labelling and in vivo calcium imaging of bone marrow-innervating dorsal root ganglia (DRG) neurons to determine the contribution of these cells in the maintenance of CIBP. We found a majority of femoral bone afferent cell bodies in L3 dorsal root ganglia (DRG) that also express the sodium channel subtype Nav1.8-a marker of nociceptive neurons-and lack expression of parvalbumin-a marker for proprioceptive primary afferents. Surprisingly, the response properties of bone marrow afferents to both increased intraosseous pressure and acid were unchanged by the presence of cancer. On the other hand, we found increased excitability and polymodality of cutaneous afferents innervating the ipsilateral paw in cancer bearing animals, as well as a behavioural phenotype that suggests changes at the level of the DRG contribute to secondary hypersensitivity. This study demonstrates that cutaneous afferents at distant sites from the tumour bearing tissue contribute to mechanical hypersensitivity, highlighting these cells as targets for analgesia.

Sensory neuron-derived NaV1.7 contributes to dorsal horn neuron excitability.

Expression of the voltage-gated sodium channel NaV1.7 in sensory neurons is required for pain sensation. We examined the role of NaV1.7 in the dorsal horn of the spinal cord using an epitope-tagged NaV1.7 knock-in mouse. Immuno-electron microscopy showed the presence of NaV1.7 in dendrites of superficial dorsal horn neurons, despite the absence of mRNA. Rhizotomy of L5 afferent nerves lowered the levels of NaV1.7 in the dorsal horn. Peripheral nervous system-specific NaV1.7 null mutant mice showed central deficits, with lamina II dorsal horn tonic firing neurons more than halved and single spiking neurons more than doubled. NaV1.7 blocker PF05089771 diminished excitability in dorsal horn neurons but had no effect on NaV1.7 null mutant mice. These data demonstrate an unsuspected functional role of primary afferent neuron-generated NaV1.7 in dorsal horn neurons and an expression pattern that would not be predicted by transcriptomic analysis.

Noninvasive Mechanical Joint Loading as an Alternative Model for Osteoarthritic Pain.

OBJECTIVE: Mechanisms responsible for osteoarthritic (OA) pain remain poorly understood, and current analgesic therapies are often insufficient. This study was undertaken to characterize and pharmacologically test the pain phenotype of a noninvasive mechanical joint loading model of OA, thus providing an alternative murine model for OA pain. METHODS: The right knees of 12-week-old male C57BL/6 mice were loaded at 9N or 11N (40 cycles, 3 times per week for 2 weeks). Behavioral measurements of limb disuse and mechanical and thermal hypersensitivity were acquired before mechanical joint loading and monitored for 6 weeks postloading. The severity of articular cartilage lesions was determined postmortem with the Osteoarthritis Research Society International scoring system. To assess efficacy of various treatments for pain, 9N-loaded mice were treated for 4 weeks with diclofenac (10 mg/kg), gabapentin (100 mg/kg), or anti-nerve growth factor (anti-NGF) (3 mg/kg). RESULTS: Mechanical hypersensitivity and weight bearing worsened significantly in 9N-loaded mice (n = 8) and 11N-loaded mice (n = 8) 2 weeks postloading, compared to baseline values and nonloaded controls. Maximum OA scores of ipsilateral knees confirmed increased cartilage lesions in 9N-loaded mice (mean ± SEM 2.8 ± 0.2; P < 0.001) and 11N-loaded mice (5.3 ± 0.3; P < 0.001), compared to nonloaded controls (1.0 ± 0.0). Gabapentin and diclofenac restored pain behaviors to baseline values after 2 weeks of daily treatment, and gabapentin was more effective than diclofenac. A single injection of anti-NGF alleviated nociception 2 days after treatment and remained effective for 2 weeks, with a second dose inducing stronger and more prolonged analgesia. CONCLUSION: Our findings show that mechanical joint loading induces OA lesions in mice and a robust pain phenotype that can be reversed using analgesics known to alleviate OA pain in patients. This establishes the use of mechanical joint loading as an alternative model for the study of OA pain.

Distinct transcriptional responses of mouse sensory neurons in models of human chronic pain conditions.

Background: Sensory neurons play an essential role in almost all pain conditions, and have recently been classified into distinct subsets on the basis of their transcriptomes. Here we have analysed alterations in dorsal root ganglia (DRG) gene expression using microarrays in mouse models related to human chronic pain. Methods: Six different pain models were studied in male C57BL/6J mice: (1) bone cancer pain using cancer cell injection in the intramedullary space of the femur; (2) neuropathic pain using partial sciatic nerve ligation; (3) osteoarthritis pain using mechanical joint loading; (4) chemotherapy-induced pain with oxaliplatin; (5) chronic muscle pain using hyperalgesic priming; and (6) inflammatory pain using intraplantar complete Freund's adjuvant. Microarray analyses were performed using RNA isolated from dorsal root ganglia and compared to sham/vehicle treated controls. Results: Differentially expressed genes (DEGs) were identified. Known and previously unreported genes were found to be dysregulated in each pain model. The transcriptomic profiles for each model were compared and expression profiles of DEGs within subsets of DRG neuronal populations were analysed to determine whether specific neuronal subsets could be linked to each of the pain models.  Conclusions: Each pain model exhibits a unique set of altered transcripts implying distinct cellular responses to different painful stimuli. No simple direct link between genetically distinct sets of neurons and particular pain models could be discerned.

Putative Promoter Motif Analyses Reinforce the Evolutionary Relationships Among Faustoviruses, Kaumoebavirus, and Asfarvirus.

Putative promoter motifs have been described in viruses belonging to the nucleocytoplasmic large DNA viruses (NCLDVs) group; however, few studies have been conducted to search for promoter sequences in newly discovered amoebal giant viruses. Faustovirus and kaumoebavirus are two Asfarviridae-related giant viruses belonging to the NCLDVs group. The phylogenetic relationships among these viruses led us to investigate if the promoter regions previously identified in the asfarvirus genome could be shared by its amoebal virus relatives. Previous studies demonstrated the role of A/T-rich motifs as promoters of asfarvirus. In this study, we reinforce the importance of A/T rich motifs in asfarvirus and show that the TATTT and TATATA motifs are also shared in abundance by faustovirus and kaumoebavirus. Here, we demonstrate that TATTT and TATATA are mostly present in faustovirus and kaumoebavirus genomic intergenic regions (IRs) and that they are widely distributed at 0 to -100 bp upstream to the start codons. We observed that putative promoter motifs are present as one to dozens of repetitions in IRs of faustovirus, kaumoebavirus, and asfarvirus, which is similar to that described previously for marseilleviruses. Furthermore, the motifs were found in most of the upstream regions of the core genes of faustovirus, kaumoebavirus, and asfarvirus, which suggests that the motifs could already be present in the ancestor of these viruses before the irradiation of this group. Our work provides an in-depth analysis of the putative promoter motifs present in asfarvirus, kaumoebavirus, and faustovirus, which reinforces the relationship among these viruses.

Genome Characterization of the First Mimiviruses of Lineage C Isolated in Brazil.

The family Mimiviridae, comprised by giant DNA viruses, has been increasingly studied since the isolation of the Acanthamoeba polyphaga mimivirus (APMV), in 2003. In this work, we describe the genome analysis of two new mimiviruses, each isolated from a distinct Brazilian environment. Furthermore, for the first time, we are reporting the genomic characterization of mimiviruses of group C in Brazil (Br-mimiC), where a predominance of mimiviruses from group A has been previously reported. The genomes of the Br-mimiC isolates Mimivirus gilmour (MVGM) and Mimivirus golden (MVGD) are composed of double-stranded DNA molecules of ∼1.2 Mb, each encoding more than 1,100 open reading frames. Genome functional annotations highlighted the presence of mimivirus group C hallmark genes, such as the set of seven aminoacyl-tRNA synthetases. However, the set of tRNA encoded by the Br-mimiC was distinct from those of other group C mimiviruses. Differences could also be observed in a genome synteny analysis, which demonstrated the presence of inversions and loci translocations at both extremities of Br-mimiC genomes. Both phylogenetic and phyletic analyses corroborate previous results, undoubtedly grouping the new Brazilian isolates into mimivirus group C. Finally, an updated pan-genome analysis of genus Mimivirus was performed including all new genomes available until the present moment. This last analysis showed a slight increase in the number of clusters of orthologous groups of proteins among mimiviruses of group A, with a larger increase after addition of sequences from mimiviruses of groups B and C, as well as a plateau tendency after the inclusion of the last four mimiviruses of group C, including the Br-mimiC isolates. Future prospective studies will help us to understand the genetic diversity among mimiviruses.

In vivo characterization of distinct modality-specific subsets of somatosensory neurons using GCaMP.

Mechanistic insights into pain pathways are essential for a rational approach to treating this vast and increasing clinical problem. Sensory neurons that respond to tissue damage (nociceptors) may evoke pain sensations and are typically classified on the basis of action potential velocity. Electrophysiological studies have suggested that most of the C-fiber nociceptors are polymodal, responding to a variety of insults. In contrast, gene deletion studies in the sensory neurons of transgenic mice have frequently resulted in modality-specific deficits. We have used an in vivo imaging approach using the genetically encoded fluorescent calcium indicator GCaMP to study the activity of dorsal root ganglion sensory neurons in live animals challenged with painful stimuli. Using this approach, we can visualize spatially distinct neuronal responses and find that >85% of responsive dorsal root ganglion neurons are modality-specific, responding to either noxious mechanical, cold, or heat stimuli. These observations are mirrored in behavioral studies of transgenic mice. For example, deleting sodium channel Nav1.8 silences mechanical- but not heat-sensing sensory neurons, consistent with behavioral deficits. In contrast, primary cultures of axotomized sensory neurons show high levels of polymodality. After intraplantar treatment with prostaglandin E2, neurons in vivo respond more intensely to noxious thermal and mechanical stimuli, and additional neurons (silent nociceptors) are unmasked. Together, these studies define polymodality as an infrequent feature of nociceptive neurons in normal animals.

Mechanisms Underlying the Scratching Behavior Induced by the Activation of Proteinase-Activated Receptor-4 in Mice.

A role for proteinase-activated receptor-4 (PAR-4) was recently suggested in itch sensation. Here, we investigated the mechanisms underlying the pruriceptive actions of the selective PAR-4 agonist AYPGKF-NH2 (AYP) in mice. Dorsal intradermal (i.d.) administration of AYP elicited intense scratching behavior in mice, which was prevented by the selective PAR-4 antagonist (pepducin P4pal-10). PAR-4 was found to be coexpressed in 32% of tryptase-positive skin mast cells, and AYP caused a 2-fold increase in mast cell degranulation. However, neither the treatment with cromolyn nor the deficiency of mast cells (WBB6F1-Kit(W/Wv) mice) was able to affect AYP-induced itch. PAR-4 was also found on gastrin-releasing peptide (GRP)-positive neurons (pruriceptive fibers), and AYP-induced itch was reduced by the selective GRP receptor antagonist RC-3095. In addition, AYP evoked calcium influx in ∼1.5% of cultured DRG neurons also sensitive to TRPV1 (capsaicin) and/or TRPA1 (AITC) agonists. Importantly, AYP-induced itch was reduced by treatment with either the selective TRPV1 (SB366791), TRPA1 (HC-030031), or NK1 (FK888) receptor antagonists. However, genetic loss of TRPV1, but not of TRPA1, diminished AYP-induced calcium influx in DRG neurons and the scratching behavior in mice. These findings provide evidence that PAR-4 activation by AYP causes pruriceptive itch in mice via a TRPV1/TRPA1-dependent mechanism.

Acanthamoeba polyphaga mimivirus stability in environmental and clinical substrates: implications for virus detection and isolation.

Viruses are extremely diverse and abundant and are present in countless environments. Giant viruses of the Megavirales order have emerged as a fascinating research topic for virologists around the world. As evidence of their ubiquity and ecological impact, mimiviruses have been found in multiple environmental samples. However, isolation of these viruses from environmental samples is inefficient, mainly due to methodological limitations and lack of information regarding the interactions between viruses and substrates. In this work, we demonstrate the long-lasting stability of mimivirus in environmental (freshwater and saline water) and hospital (ventilator plastic device tube) substrates, showing the detection of infectious particles after more than 9 months. In addition, an enrichment protocol was implemented that remarkably increased mimivirus detection from all tested substrates, including field tests. Moreover, biological, morphological and genetic tests revealed that the enrichment protocol maintained mimivirus particle integrity. In conclusion, our work demonstrated the stability of APMV in samples of environmental and health interest and proposed a reliable and easy protocol to improve giant virus isolation. The data presented here can guide future giant virus detection and isolation studies.

Characterization of a new Vaccinia virus isolate reveals the C23L gene as a putative genetic marker for autochthonous Group 1 Brazilian Vaccinia virus.

Since 1999, several Vaccinia virus (VACV) isolates, the etiological agents of bovine vaccinia (BV), have been frequently isolated and characterized with various biological and molecular methods. The results from these approaches have grouped these VACV isolates into two different clusters. This dichotomy has elicited debates surrounding the origin of the Brazilian VACV and its epidemiological significance. To ascertain vital information to settle these debates, we and other research groups have made efforts to identify molecular markers to discriminate VACV from other viruses of the genus Orthopoxvirus (OPV) and other VACV-BR groups. In this way, some genes have been identified as useful markers to discriminate between the VACV-BR groups. However, new markers are needed to infer ancestry and to correlate each sample or group with its unique epidemiological and biological features. The aims of this work were to characterize a new VACV isolate (VACV DMTV-2005) molecularly and biologically using conserved and non-conserved gene analyses for phylogenetic inference and to search for new genes that would elucidate the VACV-BR dichotomy. The VACV DMTV-2005 isolate reported in this study is biologically and phylogenetically clustered with other strains of Group 1 VACV-BR, the most prevalent VACV group that was isolated during the bovine vaccinia outbreaks in Brazil. Sequence analysis of C23L, the gene that encodes for the CC-chemokine-binding protein, revealed a ten-nucleotide deletion, which is a new Group 1 Brazilian VACV genetic marker. This deletion in the C23L open reading frame produces a premature stop-codon that is shared by all Group 1 VACV-BR strains and may also reflect the VACV-BR dichotomy; the deletion can also be considered to be a putative genetic marker for non-virulent Brazilian VACV isolates and may be used for the detection and molecular characterization of new isolates.